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Gene mutation analysis of 175 Chinese patients with early-onset epileptic encephalopathy

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Clinical Genetics
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To investigate the genetic characteristics and clinical features of a cohort of Chinese patients with early-onset epileptic encephalopathies (EOEEs). Targeted next-generation sequencing (NGS), focusing on 17 genes, was performed on 175 Chinese patients with EOEEs to screen gene mutations. The mutation rate was 32% (56/175). All mutations were de novo and heterozygous, including 41 novel and 15 reported mutations. Patients with cyclin-dependent kinase-like 5 (CDKL5) gene mutation accounted for the largest proportion-13.1% (23/175). All patients with CDKL5 mutation presented severe psychomotor developmental delay and refractory seizures. The female patients presented obvious Rett-like features, which were not observed in male patients. Potassium channel, voltage gated KQT-like subfamily Q, member 2(KCNQ2) gene mutations were detected in 13 patients. Patients with this mutation presented with early seizure onset within the first week after birth. Valproate (VPA), levetiracetam (LEV) and topiramate (TPM) were effective in most patients. Patients with specific gene mutations presented some unique clinical features, but not always. Many genes are involved in EOEEs. Targeted NGS showed a high diagnostic yield in patients with EOEEs. These findings provide useful insights for recommending treatment of gene-associated EOEEs using antiepileptic drugs.
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Clin Genet 2017: 91: 717–724
Printed in Singapore. All rights reserved
CLINICAL GENETICS
doi: 10.1111/cg e. 12901
Original Article
Gene mutation analysis of 175 Chinese patients
with early-onset epileptic encephalopathy
Zhang Q., Li J., Zhao Y., Bao X., Wei L., Wang J. Gene mutation analysis of
175 Chinese patients with early-onset epileptic encephalopathy.
Clin Genet 2017: 91: 717724. © 2016 The Authors. Clinical Genetics
published by John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.,
2016
The aim of the study is to investigate the genetic characteristics and clinical
features of a cohort of Chinese patients with early-onset epileptic
encephalopathies (EOEEs). Targeted next-generation sequencing (NGS),
focusing on 17 genes, was performed on 175 Chinese patients with EOEEs
to screen gene mutations. The mutation rate was 32% (56/175). All
mutations were de novo and heterozygous, including 41 novel and 15
reported mutations. Patients with cyclin-dependent kinase-like 5 (CDKL5)
gene mutation accounted for the largest proportion, 13.1% (23/175). All
patients with CDKL5 mutation presented severe psychomotor developmental
delay and refractory seizures. The female patients presented obvious
Rett-like features, which were not observed in male patients. Potassium
channel, voltage-gated KQT-like subfamily Q, member 2(KCNQ2)gene
mutations were detected in 13 patients. Patients with this mutation presented
with early seizure onset within the rst week after birth. Valproate (VPA),
levetiracetam (LEV) and topiramate (TPM) were effective in most patients.
Patients with specic gene mutations presented some unique clinical
features, but not always. Many genes are involved in EOEEs. Targeted NGS
showed a high diagnostic yield in patients with EOEEs. These ndings
provide useful insights for recommending treatment of gene-associated
EOEEs using antiepileptic drugs.
Conictofinterest
None of the authors has any conict of interest to disclose.
Q. Zhanga,,J.Li
b,,Y.Zhao
a,
X. Baoa,L.Wei
band J. Wanga
aDepartment of Pediatrics, Peking
University First Hospital, Beijing, China
and bCenter for Bioinformatics, State Key
Laboratory of Protein and Plant Gene
Research, School of Life Sciences,
Peking University, Beijing, China
These two authors contributed equally
to this study.
Key words: CDKL5 – early-onset
epileptic encephalopathy – KCNQ2
recommending treatment – targeted
next-generation sequencing
Corresponding authors: Xinhua Bao,
No 1, Xi’anmen, Dis Xichengqu, Beijing,
China.
Tel.: 010 83573238
fax: +010 8357 3238;
e-mail: zwhang@pku.edu.cn
and
Liping Wei, Peking University, Room
307, Wangkezhen Building, 5th
Yiheyuan Road, Beijing 100871, China.
Tel : +010-62755206
fax: +010-62755206
e-mail:weilp@mail.cbi.pku.edu.cn
Received 26 July 2016, revised and
accepted for publication 20 October
2016
Early-onset epileptic encephalopathies (EOEEs) or early
infantile epileptic encephalopathies (EIEEs) include a
series of epileptic encephalopathies, which is character-
ized by seizures onset before 6 months of age. EOEEs
have three main features: refractory seizures, severe elec-
troencephalography (EEG) abnormalities, and develop-
mental delay (DD) or intellectual disability (ID) (1).
EOEEs include some syndromes such as Ohtahara syn-
drome (OS), West syndrome (WS), early myoclonic
encephalopathy, malignant migrating focal seizures of
infancy (MMFSI), and Dravet syndrome (DS) as well
as non-specic epileptic encephalopathy (2). The eti-
ologies of this disease are complex and diverse. Beside
metabolic and structural abnormalities, genetic factors
play an important role in the pathogenesis of epilepsy
(3). Many genes related to epilepsy have been detected,
such as SCNIA,CDKL5,andALDH7A1 (4). However, it
is very time-consuming and laborious to perform conven-
tional polymerase chain reaction (PCR)-Sanger sequenc-
ing of the gene mutation responsible for the disease.
Next-generation sequencing (NGS) and whole exome
sequencing allow the analysis of a variety of genes
© 2016 The Authors. Clinical Genetics published by John Wiley & Sons A/S. Published by John Wiley & Sons Ltd. 717
This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and
reproduction in any medium, provided the original work is properly cited.
Zhang et al.
simultaneously, which is very useful in large sample
analysis or multi-gene analysis. In this study, targeted
NGS was performed to investigate 17 candidate genes
(Table 1) related to epilepsy in 175 Chinese children with
EOEEs, to identify the genetic characteristics and clini-
cal features of patients with EOEEs.
Methods
Patients
A total of 175 Chinese patients with EOEEs, including
96 females and 79 males, referred to Peking university
rst hospital between January 2012 and December 2015,
were recruited after informed consent was obtained from
their parents. All patients fullled the following crite-
ria: seizure onset within 6 months of life; severe EEG
abnormalities; DD or ID; and no identiable immediate
or remote cause; no metabolic or mitochondrial disor-
ders. This cohort of patients included 85 patients with
WS, 19 patients with Hanefeld variant of Rett syndrome
(Han-RTT), 17 patients with OS, 6 patients with MMFSI,
and 48 patients with unknown epileptic syndrome. All
patients met the diagnosis criteria of EOEEs. MECP2
mutations were negative in all patients with Han-RTT.
Clinical information, including clinical manifestation,
EEG, magnetic resonance imaging (MRI), and family
history were collected. Patients were followed up by tele-
phone or outpatient visits.
Targeted NGS
Pathogenic genes have been identied in 18 subtypes of
EOEEs according to the genetic classication updated in
2012. ‘EIEE’ was used as the search keyword to search
the ‘Online Mendelian Inheritance in Man, OMIM’ web-
site to nd the genetic information of EOEEs. Using
‘EIEE’ and ‘EOEE’ as keywords, ‘GENETICS EIEE’,
and ‘AND GENETICS EOEE’ as the search type to
search all related documents in the PubMed database,
17 genes (Table 1) were selected as candidate genes that
may lead to the pathogenesis of early-onset epilepsy.
Patients diagnosed with DS were not recruited in our
study because its clinical manifestation is easily identi-
ed and SCN1A is the main pathogenic gene of DS.
The AmpliSeqlibrary was prepared following the mod-
ied Ion AmpliSeqlibrary preparation protocol (Pub No.
MAN0006735). The Illumina adapters were used instead
of standard Ion adapters for higher throughput and lower
cost. Two rounds of enrich PCR instead of one were
performed to increase the efciency of the elongation
step during the PCR when adding the barcode on the
library. Detailed steps are described in the Supporting
information. The libraries were quantied by qPCR and
pooled together according to the molecular concentra-
tion. The pooled library was sequenced on Illumina
HiSeq 2500 (CA, USA), generating approximately 1M
100-bp pair-end reads for each sample.
Fast QC v0.10.1 was used to check the quality of
the reads and bwa0.7.12-r1039 to align the reads to the
hg19 genome, producing a le in BAM format sorted by
coordinates. Local realignment around indels and base
quality score recombination by GATK v3.2 were per-
formed on the BAM le for pre-processing. We used
Unied Genotyper and Hapolotype Caller in GATK
v3.2 to call variants. Rare mutations, whose popula-
tion frequency were less than 1%, were ltered accord-
ing to the 1000 Genomes data, ESP6500 population
data, and ExAC data. Rare variants were annotated to
gene and protein change by Annovar July2015 with
the RefSeq Gene dataset. Reported pathogenic muta-
tions in HGMD Professional database and PubMed were
marked, while the pathogenicity of other rare mutations
was annotated by Mutation Taster. We performed valida-
tion and parental origin analyses for these variations by
PCR-Sanger sequencing and conrmed causative muta-
tions according to parental origin of the variations and
clinical features of the patients.
Results
Heterozygous mutations in seven genes were detected in
56 patients, including 41 novel mutations and 15 reported
mutations. All mutations were predicted to be ‘disease
causing’ by Mutation Taster. The gene mutation rate
was 32% (56/175). CDKL5 mutations were detected in
23 patients, representing the largest proportion (13.1%;
23/175). The second most detected mutations were
KCNQ2 mutations, detected in 13 cases. The third one
was KCNT1 mutation, identied in six cases. Other
detected gene mutations included those in the STXBP1
gene in ve patients, SCN2A in four patients, SCN8A in
three patients, and SLC2A1 in two patients. Each patient
had a unique mutation, except for twin sisters presenting
with the same CDKL5 mutation. The clinical features and
mutations of the patients are summarized in Table 2.
CDKL5 mutations included 6 missense mutations, 3
splicing site mutations, and 14 mutations, which lead to
a premature termination codon. All mutations were de
novo mutations. None of the parents carried the same
mutation. Among the 23 patients with CDKL5 muta-
tions, 20 were females accounting for 20.8% (20/96)
of female patients with EIEE and only 3 were males
(3.8%, 3/79). Out of the 23 patients who carried a
CDKL5 mutation, 11 females (57.9%, 11/19) were diag-
nosed with Han-RTT, followed by 3 males and 7 females
(8.5%, 10/85) with WS, and 2 females (4.2%, 2/48)
with non-syndromic epilepsy. All 23 patients suffered
early-onset seizures before 4 months of age (from 10 to
100 days after birth).Various seizure types presented in
the course of the disease, including epileptic spasms, par-
tial seizures, myoclonic seizures, and tonic seizures. The
initial seizure types of the patients were partial seizures
in 20 cases and tonic seizures in 3 cases. Epileptic spasms
developed in 22 patients later in life, the rest one only pre-
sented with partial spasms and tonic seizures. The age of
epileptic spasm onset ranged from 10 to 100 days after
birth. Hypsarrhythmia was identied on EEG records in
14 patients. The epilepsy in patients with CDKL5 gene
mutations was intractable and resistant to antiepilep-
tic drugs (AEDs). Eleven female patients showed some
Rett-like features, such as microcephaly, limited hand
718
Gene mutation analysis of Chinese patients with EOEEs
Table 1. List of 17 candidate genes
Gene name Abbreviation Gene number Gene location
Cyclin-dependent kinase-like 5 CDKL5 NM_001037343 chrX:18,442,188-18,653,629
Aristaless-related homeobox ARX NM_139058 chrX:25,003,694-25,015,948
Syntaxin-binding protein 1 STXBP1 NM_001032221 chr9:127,612,283-127,692,716
spectrin, alpha, non-erythrocytic 1 SPTAN1 NM_003127 chr9:128,552,558-128,633,662
solute carrier family 2 SLC2A1 NM_006516 chr1:42,925,375-42,959,173
solute carrier family 25 SLC25A22 NM_001191061 chr11:790,475-798,333
sodium channel, voltage gated, type I alpha subunit SCN1A NM_006920 chr2:165,989,163-166,073,639
potassium channel, voltage-gated KQT-like subfamily Q, member 2 KCNQ2 NM_172107 chr20:63,406,137-63,472,590
sodium channel, voltage gated, type II alpha subunit SCN2A NM_001040142 chr2:165,239,402-165,392,308
Cdc42 guanine nucleotide exchange factor (GEF) 9 ARHGEF9 NM_001173479 chrX:63,636,331-63,785,524
protocadherin 19 PCDH19 NM_001105243 chrX:100,291,646-100,408,597
polynucleotide kinase 3-phosphatase PNKP NM_007254 chr19:49,861,204-49,867,886
phospholipase C, beta 1 PLCB1 NM_015192 chr20:8,132,265-8,884,903
sodium channel, voltage gated, type VIII alpha subunit SCN8A NM_014191 chr12:51,591,236-51,812,864
potassium channel, subfamily T, member 1 KCNT1 NM_020822 chr9:135,702,192-135,793,103
ST3 beta-galactoside alpha-2,3-sialyltransferase 3 ST3GAL3 NM_006279 chr1:43,707,547-43,931,159
aldehyde dehydrogenase 7 family, member A1 ALDH7A1 NM_001182 chr5:126,541,841-126,595,418
skills, and stereotypic hand movements, which were
not detected in patients with WS and non-syndromic
epilepsy, and the three male patients. One patient died
of severe pulmonary infection at 2 years of age.
KCNQ2 gene mutations were identied in 13 patients,
including 12 missense mutations and 1 micro-deletion
mutation. All mutations were de novo mutations. Four
mutations were located in exon 5, and six mutations
were located in exon 6. The other three mutations were
located in exons 4, 12, and 13, respectively. In our cohort,
KCNQ2 mutation rate was 7.4% (13/175). Among the
13 cases, 6 were males and 7 were females. Eight cases
(9.4%, 8/85) were diagnosed with WS, two cases (11.8%,
2/17) with OS, and three cases (6.25%, 3/48) with
non-syndromic epileptic encephalopathy. All patients
presented with severe DD. Twelve patients had seizure
onset in the rst week of life, the rest one had seizure
onset at 9 days of life. The semiology of 11 patients
was characterized by tonic or asymmetric tonic seizures,
accompanied by cyanosis. The other seizure types were
tonic seizures, epileptic spasms, partial seizures, and
atypical absence seizures. EEG showed hypsarrhythmia
in 11 patients and frequent multifocal epileptic activity
in the other 2 patients. Seven patients were completely
seizure free after taking AEDs at 322 months of age.
One patient had two periods of seizure freedom. Brain
MRI, blood and urine amino acid and organic acid were
normal in all patients.
Clinical seizure freedom was achieved in seven
patients after AEDs therapy (detail in Fig. 1). The age
of seizure freedom ranged from 3 to 22 months of age.
Two patients were treated with sodium valproate (VPA)
and levetiracetam (LEV). One patient was treated with
VPA, LEV, and topiramate (TPM). One patient was
treated with TPM, LEV, and clonazepam (CZP). One
was treated with VPA, TPM, and CZP. One patient
was treated with LEV and TPM. The last one was
treated with carbamazepine (CBZ). Epilepsy in the
other six patients was intractable, but seizure frequency
was reduced in four patients to 14 times per month.
Only one patient had no signicant improvement after
multiple AED treatments. However, one case among
them had two periods of clinically seizure freedom:
for 11 months by treatment with LEV and vigabatrin
(VGB) and for 5 months with VPA and TPM treatment.
Hypsarrhythmia was ever presented in EEG among 11
patients, then changed to multifocal epileptic activity
after effective treatment, especially treated with ACTH.
All six patients with intractable seizure ever had hypsar-
rhythmia. MRI showed myelination delay in the cerebral
white matter in two patients and thinning of the corpus
callosum in two of them.
Missense mutations of KCNT1 were detected in six
patients, including two males and four females. The age
of nal follow-up was ranging from 5 to 31 months.
Seizure onset was from 2 to 32 days after birth. All of
them were diagnosed with MMFSI. All patients with
KCNT1 gene mutation had intractable epilepsy, which
was resistant to AEDs. Seizure frequency in one case
was signicantly reduced after taking quinidine. The
seizure attacks decreased from more than 100 to 10 times
per day. Hypsarrhythmia was identied on EEG records
in four patients. All patients had severe developmen-
tal delay and refractory seizures. None of these patients
could speech or walk. In addition, frequent upper res-
piratory tract infections occurred in these patients. One
patient died of severe pulmonary infection at 1 year and
4 months of age.
STXBP1 gene mutations, including four missense and
one nonsense mutation, were detected in ve patients,
one male and four females. All patients were diagnosed
with OS (29.4%, 5/17) with severe developmental delay.
They had seizures onset within the neonatal period (from
2 to 15 days), which was refractory to AEDs.
Missense mutations of SCN2A gene were detected
in four patients, one female and three males. Three
patients were diagnosed with non-syndromic epileptic
encephalopathy, one with WS. The seizures onset in
719
Zhang et al.
Table 2. Summary of clinical features of patients and mutations
Gene EOEE_ID Gender Mutation AA change
Novel/
reported Exon Age (month) onset (day) Diagnose
Seizure
free
CDKL5 13 F 1111delC L371fsX492 N 12 24 22 West
CDKL5 32 F ISV13+1G>A N 36 10 Han-RTT –
CDKL5 93 F 1833_1834delTT H611fsX617 N 12 36 40 Han-RTT –
CDKL5 34 F ISV6+1G>A R 48 100 Han-RTT –
CDKL5 39 F 1375C>T Q459X N 12 87 30 Han-RTT –
CDKL5 45-1 F 891_892insTT F298fsX349 N 11 30 90 Han-RTT –
CDKL5 45-2aF 891_892insTT F298fsX349 N 11 30 55 Han-RTT –
CDKL5 50 M 533G>A R178Q R 8 5 60 West –
CDKL5 77 F 2360delA K787fsX802 N 16 17 30 Han-RTT –
CDKL5 68 F 234delA A78fsX112 N 5 42 40 Han-RTT –
CDKL5 1 F 1791_1792insG G597fsX610 N 12 36 40 Non-syn –
CDKL5 54 F G100T E34X N 4 13 28 West –
CDKL5 106 F 1785 delA G595 fsX615 N 12 12 21 West
CDKL5 116 M 215T>A I72N R 5 6 27 West –
CDKL5 124 F 426-430del5bpAATCA L142 fsX 145 N 7 20 40 Han-RTT
CDKL5 145 F 1245-1246delAG T415fsX417 N 12 13 60 Han-RTT –
CDKL5 162 F IVS9-1G>AN8.657West
CDKL5 159 F T260C L87S N 5 32 40 Non-syn –
CDKL5 14 F 991delA K331fsX349 N 12 9 30 West
CDKL5 148 F 683G>A G228E N 9 8.6 90 Han-RTT –
CDKL5 121 F T182C L61P N 5 72 80 West –
CDKL5 111 F C119T A40V R 5 4.5 46 West –
CDKL5 ZH M 1326-1327insT N443fsX R 12 11.2 20 West
KCNQ2 64 F c.T850C Y284H N 6 4 1 West –
KCNQ2 69 F c.A871G R291G N 6 16 1 West –
KCNQ2 70 M c.A710T Y237F N 5 51 2 West SF
KCNQ2 97 F c.G868A G290S N 6 24 3 Non-syn SF
KCNQ2 117 F c.T838C Y280H N 6 26 9 West –
KCNQ2 134 M c.G1452C E484D R 13 12 1 OS SF
KCNQ2 135 M c.1284delG Q429Rfs*5 N 12 3 2 West SF
KCNQ2 137 F c.T913C F305L N 6 2.9 5 West –
KCNQ2 133 M C.736G>C A246P N 5 19 1 West –
KCNQ2 ZYS M c.793G>A A265T R 5 28 1 West SF
KCNQ2 ZZ F c.748G>T V250L N 5 18 4 Non-syn SF
KCNQ2 CXY M c.821C>T T274M R 6 15 2 West –
KCNQ2 WXF M c.637C>T R213WB R 4 84 2 Non-syn SF
KCNT1 79 M c.C2797G R933G N 24 5 3 MMFSI –
KCNT1 91 M c.A1885G K629E N 18 31 2 MMFSI –
KCNT1 94 M c.G811T V271F R 10 29 2 MMFSI –
KCNT1 107 F c.G1283AC R428Q R 13 21 2 MMFSI –
KCNT1 108aM c.G2800A A934T R 24 16 32 MMFSI –
KCNT1 118 F c.G1421AE R474H R 15 23 28 MMFSI –
STXBP1 102 F c.C364T R122X N 6 2.4 14 OS
STXBP1 60 F c.C1439T P480L N 16 2 2 OS
STXBP1 123 F c.G875A R292H R 10 0.8 3 OS
STXBP1 131 M c.C416A P139Q N 6 3.2 3 OS
STXBP1 WJL F c.A1661G Y554C N 18 24 3 OS
SCN2A 14 F c.G781A V261M N 6 3.6 2 Non-syn –
SCN2A 67 M c.T5636C M1879T N 26 37 5 Non-syn SF
SCN2A 92 M c.G3956T R1319L N 20 13 2 Non-syn SF
SCN2A 139 M c.4364T>A I1455N N 23 1.9 2 West
SLC2A1 LCZ F c.599delA Q200RfsX N 5 6 33 Glut1-DS –
SLC2A1 CYF M c.1372C>T R458W R 10 9 120 Glut1-DS SF
SCN8A 83 M c.C5614T R1872W R 27 12 105 Non-syn –
SCN8A GSY M c.694T>C S232P N 5 12 61 Non-syn SF
SCN8A DMY M c.2549G>A R850Q N 16 24 90 Non-syn SF
Glut1-DS: glucose transporter 1 deficiency syndrome; Han-RTT: Hanefeld variant of Rett syndrome; MMFSI: malignant migrating
focal seizures of infancy; N (for novel)/R (for reported); Non-syn; non-specific epileptic encephalopathy; OS: Ohtahara syndrome; SF:
seizure free; West: west syndrome; –: negtive.
aPatient died of died of severe pulmonary infection.
720
Gene mutation analysis of Chinese patients with EOEEs
Fig. 1 . Antiepileptic drugs (AEDs) prole of seven patients who became
seizure-free. The horizontal axis represents the ID of the seven patients.
CBZ, carbamazepine; LEV, levetiracetam; CZP, clonazepam; VPA,
valproate; TPM, topiramate.
these patients was within the rst week of life (from
2 to 5 days after birth). Seizure freedom was achieved
in two patients after AED treatment. One patient was
treated with VPA and TPM, another one with VPA
and CBZ. Seizure frequency was remarkably reduced in
one patient treated with VPA and CBZ. The other had
intractable seizure. All patients presented with severe
developmental delay.
Missense mutations of SCN8A were detected in three
patients. All patients were males. The seizure onset
was within 3 months of age (from 2 to 3 months after
birth). Seizure types included partial seizures, myoclonic
seizures, and partial seizure, secondarily generalized.
Two patients were seizure free after treatment with
AEDs, one with VPA and LTG and the other one with
VPA, LEV and oxcarbazepine.
SLC2A1 gene mutations, including one missense muta-
tion and one micro-deletion, were detected in two
patients. One was a female and the other a male. Their
phenotype included paroxysmal exercise-induced dyski-
nesia and epilepsy, non-epileptic allelic variants such as
confusion, lethargy, or somnolence, and total body paral-
ysis. These two patients had seizure onset at 33 days and
4 months of age. The seizure type in the two patients was
partial seizures. Both were diagnosed with glucose trans-
porter 1 deciency syndrome (GLUT1-DS), as these
neurologic signs could be inuenced by factors such as
fasting or fatigue. In addition, the glucose levels in the
cerebrospinal uid of these two patients were 1.74 and
2.01 mmol/L, respectively. These two patients presented
with mental and development retardation. The male
patient responded well to frequent meals with snacks.
The ketogenic diet was introduced to the female patient,
but her parents gave up this treatment shortly because of
her intolerance to the food and the lack of response to the
ketogenic diet.
Discussion
The detection rate of targeted NGS in our study was 32%
(56/175). This study illustrates the diagnostic efciency
of complementary genetic approaches in children with
EOEEs of unknown etiology. Our ndings suggest that
some unexplained sporadic EOEEs cases involve de novo
pathogenic mutations (missense/nonsense/frameshift
mutations).
CDKL5-related diseases include Han-RTT, X-linked
infantile spasms, EIEE-2, autism spectrum disorders,
Rett-like syndrome, and Angleman-like syndrome
(57). In this study, the CDKL5 mutational rate was
13.1% (23/175), which is similar to that of previous
studies (from 8% to 28%) (6, 8, 9). CDKL5 mutations
were predominantly detected in females. Core symp-
toms of the patients’ with CDKL5 mutations included
epilepsy, severe psychomotor developmental delay,
mental retardation, hypotonia, and slow growth in head
circumference. Partial seizures and tonic seizures are
the initial seizure types. Epileptic spasms are a com-
mon seizure type in the later course of the disease.
Other seizure types included myoclonic seizures, tonic
seizures, and atypical absence. Hypsarrhythmia or atyp-
ical hypsarrhythmia were detected in 14 patients. The
epileptic events were resistant to all antiepileptic treat-
ments. None of these children could talk or walk. Twelve
patients could sit independently, and the acquired time
was delayed from 10 to 24 months in seven patients.
Although all patients showed some autistic symptoms
such as poor eye contact and limited interest, only
11 female patients fullled the criteria of Han-RTT;
showing some Rett-like features such as stereotypic
hand movements and microcephaly. To date, more than
20 male patients with CDKL5 mutations have been
reported (10, 11). Compared with female patients, males
presented more severe phenotypes, manifested as severe
mental and motor retardation. The limited hand-use and
stereotyped movements were less signicantly detected
in male than in female patients (11). The age of the three
male patients with CDKL5 mutations ranged from 5 to
11 months in our study. The seizure onset was from 20
to 60 days after birth. None of them could lift their heads
or acquired sitting or walking skills. Rett-like features
such as hand stereotypies were not obvious in the three
male patients.
The KCNQ2 gene, encoding a voltage-gated potassium
(Kv7.2) channel, is responsible for about 10% of EOEEs
with neonatal onset (12, 13). Kv7 channels assemble as
tetramers, with each subunit displaying a core domain
formed by six transmembrane segments (S1– S6), and
cytoplasmic N- and C-termini. Within each subunit,
the S1S4 segments form the voltage-sensing domain
arranged symmetrically around the pore (Fig. 2). More
than 50 KCNQ2 mutations associated with epileptic
encephalopathy were reported (1416). The hallmark of
this disorder is the onset of refractory seizures within
the rst week of life. Motor and cognitive decits
are obvious from birth. The common seizure type is
tonic seizures and is often asymmetric with ocular
symptoms and apnea. Some patients become seizure free
after several months to years (ranging from 2 months
of age to adolescence) (13). At onset, EEG shows
multifocal epileptic form of activity or a hypsarrhythmia
pattern (17). Brain imaging may reveal hypoplasia of
the corpus callosum, hyperintensity in the basal ganglia,
and diffuse hypomyelination (18). In our study, KCNQ2
mutations were detected in 13 patients (7.4%, 13/175).
721
Zhang et al.
All patients with KCNQ2 mutations presented with
neonatal seizures and severe developmental impairment.
Seizures started within 9 days of age. The initial seizure
types were tonic seizures or asymmetric tonic seizures.
The effective AEDs were VPA, LEV, TPM and CBZ
(Fig. 1). Pisano et al. reported that CBZ and phenytoin
(PHT) were the rst-line treatment in patients with
KCNQ2 encephalopathy (17). CBZ and PHT were not
recommended medication for 10 cases among these
13 patients presented with spasm seizures. However,
LEV, TPM, VPA were effective in many patients in the
study of Pisano et al. (17) VPA, LEV and TPM were
recommended drugs for the treatment to patients with
KCNQ2 mutations, especial when their seizure type was
spasm.
An important phenomenon was observed in this
study. Mutations in six patients with uncontrolled
epilepsy were located in the pore-forming S5 and S6
regions of Kv7.2 channel (Fig. 2). Haploinsufciency
and dominant-negative effect are the main patho-
mechanism underpinning the severe phenotype of
KCNQ2-related encephalopathy. KCNQ2 mutations
decrease the inhibitory potassium current across cell
membranes (19). All these KCNQ2 mutations can lead
to haploinsufciency. KCNQ2 mutations of the six
patients with intractable seizure were all located in the
area of the pore-loop between the S5 and S6 regions.
The other patients with KCNQ2 mutations responded
well to AEDs. Dominant-negative effect at subthreshold
voltages may be the mechanism underlying the KCNQ2
refractory epilepsy, this need more test to conrm this
phenomenon. Orhan et al. detected that two pore muta-
tions induce a dominant-negative effect by a reduction
of the overall current amplitude yielded larger currents
(19). Amino acids in these regions may have a more
important function, explaining the more severe phe-
notype. Another hypothesis is that the binding site of
AEDs is probably located in the pore region, suggesting
that the pore mutations may directly affect the binding
site. Studies to identify a differential responsiveness of
individual mutants to drug application may serve as a
basis for further analysis of the disease mechanisms of
KV7.2-related EOEEs.
The six KCNT1 mutations were located in the KCNT1
protein (Fig. 3). KCNT1 encodes a sodium-activated
potassium channel subunit that plays an important role
in regulating neuron excitability. KCNT1 is not widely
expressed in the cortex, but is highly expressed in neu-
rons of the frontal cortex. (20) All pathogenic mutations
in KCNT1 channels result in a strong gain-of-function
phenotype. KCNT1 is identied as a cause of two
forms of early-onset epileptic disorders, autosomal dom-
inant nocturnal frontal lobe epilepsy (ADNFLE) (21)
and MMFSI (22). ADNFLE usually occurs in individ-
uals of normal intellect and its mean age of onset is
9 years (ranging from 1 to 15 years) (23). MMFSI is a
severe EOEE and the age of epilepsy onset is before
6 months of age (22). Hyperactivation of KNa channels
is the patho-mechanism of this disease. In this study,
all six patients with KCNT1 mutations met the diagnos-
tic criteria for MMFSI. The seizure onset ranged from
Table 3. Classification of mutated genes
Gene function Mutated gene Total cases
Ion channel KCNQ2,KCNT1,
SCN2A,SCN8A
26
Protein kinase CDKL5 23
Synapse STXBP1 5
Cell metabolism SLC2A1 2
2 to 32 days after birth. Video-EEG recordings showed
the specic migrating feature, presenting with ictal dis-
charges on EEG occurring in one region and migrating
to another in all patients. The prognosis of these patients
is extremely poor with intractable seizures and severe
psychomotor retardation. None of them had the abil-
ity of speak or walk. Researchers identied that quini-
dine could reverse KCNT1 gain-of-function mutation
(24, 25). One of our patients had a nearly 90% decrease
in seizure frequency after taking quinidine.
SCN8A and SCN2A have already been reported as
important genes in epileptic encephalopathy. STXBP1 is
a main pathogenic gene of OS, while SLC2A1 is asso-
ciated with GLUT1-DS. The ketogenic diet is effective
at controlling seizures in most patients with GLUT1-DS
(26). The follow-up of the two patients with SLC2A1
mutations was about 1 year. Hypoglycorrhachia was
detected in these two patients. They all fullled the cri-
teria of GLUT1-DS. One patient responded well to a fre-
quent meal supplemented with snacks, while the other
one could not tolerate the ketogenic diet and did not
respond to this treatment.
To understand the possible underlying pathology of
EOEEs, the mutated genes identied in our cohort were
divided into four groups according to gene function
(Table 3). The largest portion included genes encod-
ing ion channels (46.4%, 26/56), suggesting that ion
channels play important roles in the pathogenesis of
EOEEs. The abnormal ion transport may affect vari-
ous processes such as nerve excitation, cell proliferation,
sensory transduction, learning, and memory, resulting
in EOEEs (27, 28). Furthermore, some factors in pro-
tein kinase modulation, synapse, transcriptional regula-
tion, cell metabolism, and cellcell interaction are also
involved in the pathogenesis of EOEEs. CDKL5 is a pro-
tein kinase and cyclin-dependent kinase. CDKL5 defect
may affect the early steps of differentiation by altering
molecular processes, resulting in a delayed renement
of cortical architecture, besides impacting cytoskele-
tal modications (29). Synapse formation and normal
function are important for developmental processes that
contribute to circuit renement in the nervous system
(30). Moreover, genes related to synapse elimination and
maturation are closely related to EOEEs. However, the
understanding of the mechanisms of EOEEs is currently
limited. Further studies of the neurological dysfunctions
underlying the etiology of EOEEs are urgently needed.
Seizures, especially early-onset epilepsy, are usually
intractable and co-occurring with a poor motor and cog-
nitive prognosis. Therefore, it is very important to deter-
mine the cause of EOEEs and explore treatment options.
722
Gene mutation analysis of Chinese patients with EOEEs
Fig. 2 . Structure of the KCNQ2 (KV7.2) channel protein and mutation sites. KV7.2 has the typical structure of voltage-gated potassium channel subunits,
with intracellular N- and C-termini, six transmembrane segments (S1– S6), a voltage-sensing domain (VSD) formed by S1 S4, and the pore-loop
between S5 and S6. Red dots represent the locations of KCNQ2 mutations in easily controlled epilepsy. Blue dots represent the locations of KCNQ2
mutations in intractable epilepsy.
Fig. 3 . Model of the KCNT1 channel protein and showing locations of mutations identied in this study. The KCNT1 protein consists of six hydrophobic
transmembrane segments (S1– S6) with the pore-loop between S5 and S6. It has a large intracellular carboxy-terminal region containing tandem RCK
domains and an NAD+binding domain. Red dots represent the locations of KCNT1 mutations.
A rapid and efcient system of target capture sequenc-
ing can be applied to the comprehensive genetic analy-
sis of EOEEs. In this study, targeted NGS was used to
investigate the causative gene mutations in 175 Chinese
children with unexplained EOEEs. The causing genes
were established in 56 patients. It expanded the pheno-
type and mutation spectrum of seven genes associated
with EOEEs. With a gene detection rate of 32%, targeted
NGS is certainly considered an efcient and precise
approach to screen monogenic mutations in patients with
EOEEs. However, according to our experience, some
limitations of this approach and tips for better detec-
tion should be discussed. First, to eliminate false posi-
tive results, conventional Sanger sequencing is required
for the validation of the variations considered signicant
by targeted NGS. Secondly, targeted NGS resulted in
false negative results. In our study, the majority of cases
(68%, 119/175) remained unexplained. This observa-
tion indicates that additional candidate pathogenic genes
need to be detected in the future. We will also add
more pathogenic genes to our panel in the future. Third,
copy number variations (CNVs) were not detected in
723
Zhang et al.
our study. Thus, array comparative genomic hybridiza-
tion and multiplex ligation-dependent probe amplica-
tion were needed to detect the CNVs. In this regard, we
did not summarize the characteristics of the gene muta-
tion in patients with DS because these patients were not
recruited for our study.
In this study, we summarized the clinical features
of EOEEs patients with CDKL5,KCNQ2,andKCNT1
mutations, and determined that LEV, VPA and TPM
are the recommended drugs for patients with KCNQ2
mutations, and quinidine is a recommended drug for
patients with KCNT1 mutations. Our study indicates that
gene mutations are highly pleiotropic and can cause a
wide spectrum of seizure disorders, and the genetic study
may guide the treatment of gene-associated-epilepsy
in the future. Our study provides useful insights that
can help improve the routine diagnosis of EOEEs and
understanding of the genetic architecture of epilepsy as
well as the clinical diagnosis, treatment, and the clinical
and genetic counseling to families of patients.
Supporting Information
Additional supporting information may be found in the online
version of this article at the publisher’s web-site.
Acknowledgements
We thank the patients and their parents for their cooperation in this
study. This study was nancially supported by 985 Peking Uni-
versity and Clinical Hospital Cooperation Project (2013-1-06). We
would like to thank Editage (www.editage.cn) for English language
editing.
Ethics approval
The study was approved by Clinical Research Ethics
Committee, Peking University (NM: IRB00001052-
11087). We conrm that we have read the journal’s
position on issues involved in ethical publication and
afrm that this report is consistent with those guidelines.
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... While the mutation found in 10 (5.5%) patients was considered definitively pathogenic, the changes found in 11 (5.5%) patients were determined as likely pathogenic. In the studies conducted by Ara k Parrini E and Trump N, this rate was found to be between 19.5 and 26.6% [9][10][11] . The high number of patients observed in these studies is thought to be effective in obtaining these results. ...
... With NGS systems, faster epilepsy genes will be detected. In other studies, these rates varied between 14.5 and 41.6% [8][9][10] . West syndrome developed in four patients (25%), and LGS developed after West syndrome in two patients. ...
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... It is hypothesized that ASMs bind to the pore region of channels and that variants in this area could impair binding, necessitating functional analyses to inform treatment. Previous findings have shown that ASMs likely target the pore region of channels, suggesting that variants in this vicinity could interfere with ASM binding 25 . Interestingly, these findings include a patient with a missense variant at the same Val250 position but different in altering amino acid, c.748G > C; (p.Val250Leu) who showed a positive response to ASMs, such as VPA and levetiracetam (LEV), suggesting these drugs for individuals with KCNQ2 variants 25 . ...
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We report two patients with drug-resistant epilepsy, caused by KCNT1 mutations, who were treated with quinidine. Both mutations manifested gain of function In-vitro, showing increased current that was reduced by quinidine. One, who had epilepsy of infancy with migrating focal seizures, had 80% reduction in seizure frequency. The other, who had a novel phenotype, with severe nocturnal focal and secondary generalized seizures starting in early childhood with developmental regression, did not improve. While quinidine represents an encouraging opportunity for therapeutic benefits, our experience suggests caution in its application and supports the need to identify more targeted drugs for KCNT1 epilepsies. This article is protected by copyright. All rights reserved.
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Autosomal dominant mutations in the sodium-gated potassium channel subunit gene KCNT1 have been associated with two distinct seizure syndromes, nocturnal frontal lobe epilepsy (NFLE) and malignant migrating focal seizures of infancy (MMFSI). To further explore the phenotypic spectrum associated with KCNT1, we examined individuals affected with focal epilepsy or an epileptic encephalopathy for mutations in the gene. We identified KCNT1 mutations in 12 previously unreported patients with focal epilepsy, multifocal epilepsy, cardiac arrhythmia, and in a family with sudden unexpected death in epilepsy (SUDEP), in addition to patients with NFLE and MMFSI. In contrast to the 100% penetrance so far reported for KCNT1 mutations, we observed incomplete penetrance. It is notable that we report that the one KCNT1 mutation, p.Arg398Gln, can lead to either of the two distinct phenotypes, ADNFLE or MMFSI, even within the same family. This indicates that genotype-phenotype relationships for KCNT1 mutations are not straightforward. We demonstrate that KCNT1 mutations are highly pleiotropic and are associated with phenotypes other than ADNFLE and MMFSI. KCNT1 mutations are now associated with Ohtahara syndrome, MMFSI, and nocturnal focal epilepsy. They may also be associated with multifocal epilepsy and cardiac disturbances. Wiley Periodicals, Inc. © 2015 International League Against Epilepsy.
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Objectives To describe the antiepileptic drug (AED) treatment of patients with early infantile epileptic encephalopathy due to KCNQ2 mutations during the neonatal phase and the first year of life.Methods We identified 15 patients and reviewed the electroclinical, neuroimaging, and AED treatment data.ResultsSeizure onset was between 1 and 4 days of age with daily tonic asymmetric, focal and clonic seizures in nine patients and status epilepticus in the remaining six. Electroencephalography (EEG) showed multifocal epileptiform abnormalities in nine patients and a burst-suppression pattern in six. All patients were trialed with adequate daily doses of several AEDs before they reached seizure freedom. Six patients (40%) achieved seizure control within 2 weeks of carbamazepine (CBZ) administration and five (33%) were seizure-free with phenytoin (PHT). The last four patients (27%) were successfully treated with topiramate (TPM) (two patients), levetiracetam (LEV) (one), and a combination of LEV with TPM (one). Most patients reached seizure freedom within the first year of life and remained seizure-free thereafter. Twelve patients had moderate-to-severe developmental delay at follow-up. However, the two patients whose seizures ceased within a few days of onset showed only mild cognitive impairment.SignificanceOur findings suggest that drugs acting on sodium channels including CBZ and PHT should be considered as first-line treatment in patients with KCNQ2 encephalopathy. Voltage-gated sodium and potassium channels co-localize at the neuronal membrane. Therefore, the efficacy of drugs acting as sodium-channel blockers could be linked to their modulating effect on both channels. The type of KCNQ2 mutation might influence AED response as well as developmental outcome. Early recognition of KCNQ2 encephalopathy followed by the most appropriate and effective treatment may be important for reducing the neurodevelopmental impairment associated with this disorder.
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Epileptic encephalopathies represent a group of devastating epileptic disorders that occur early in life and are often characterized by pharmaco-resistant epilepsy, persistent severe electroencephalographic abnormalities, and cognitive dysfunction or decline. Next generation sequencing technologies have increased the speed of gene discovery tremendously. Whereas ion channel genes were long considered to be the only significant group of genes implicated in the genetic epilepsies, a growing number of non-ion-channel genes are now being identified. As a subgroup of the genetically mediated epilepsies, epileptic encephalopathies are complex and heterogeneous disorders, making diagnosis and treatment decisions difficult. Recent exome sequencing data suggest that mutations causing epileptic encephalopathies are often sporadic, typically resulting from de novo dominant mutations in a single autosomal gene, although inherited autosomal recessive and X-linked forms also exist. In this review we provide a summary of the key features of several early- and mid-childhood onset epileptic encephalopathies including Ohtahara syndrome, Dravet syndrome, Infantile spasms and Lennox Gastaut syndrome. We review the recent next generation sequencing findings that may impact treatment choices. We also describe the use of conventional and newer anti-epileptic and hormonal medications in the various syndromes based on their genetic profile. At a biological level, developments in cellular reprogramming and genome editing represent a new direction in modeling these pediatric epilepsies and could be used in the development of novel and repurposed therapies.
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Objective Mutations involving the cyclin-dependent kinase-like 5 (CDKL5) gene cause an early onset epileptic encephalopathy (EE) with severe neurologic impairment and a skewed 12:1 female-to-male ratio. To date, 18 mutations have been described in boys. We analyzed our cohort of boys with early onset EE to assess the diagnostic yield of our molecular approach.Methods We studied 74 boys who presented early onset severe seizures, including infantile spasms and developmental delay, in the setting of EE, using Sanger sequencing, next-generation sequencing (NGS) and multiplex ligation-dependent probe amplification (MLPA).ResultsWe identified alterations involving CDKL5 in four boys (5.4%) using NGS in one and MLPA in three. Three of four mutations were indicative of somatic mosaicism.SignificanceCDKL5 gene mutations accounted for 5.4% of boys with early onset EE. Somatic mosaic mutations might be even more represented than germline mutations, probably because their less deleterious effect enhances viability of the male embryo. The molecular approach used for CDKL5 screening remarkably influences the diagnostic yield in boys. Diagnosis is optimized by Sanger sequencing combined with array-based methods or MLPA; alternatively, NGS targeted resequencing designed to also detect copy number alterations, may be performed.
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